The major histocompatibility complex of humans is a cluster of genes occupying a region located on the sixth chromosome. Human leukocyte antigen ("HLA") genes are highly polymorphic and code for human leukocyte antigens whose structural variation is a major factor in influencing tissue transplantation, immunity and autoimmunity. The polymorphic HLA proteins have been designated HLA-A, -B, -C, -DR, -DQ and -DP. The HLA-A, -B, and -C proteins are described as Class I HLA proteins and are characterized by a polymorphic chain, alpha, and a nonpolymorphic chain, beta 2 microglobulin. The HLA-DR, -DQ and -DP proteins are classified as Class II HLA proteins and are comprised of two polypeptide chains, an alpha chain and a highly polymorphic beta chain. These HLA-D-region proteins are encoded by loci designated HLA-DRA, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. HLA-DRA, -DQA1 and -DPA1 are much less polymorphic than HLA-DRB1, DQB1 and -DPB1.
The proteins encoded by the polymorphic HLA loci are most commonly typed using serological methods. One of the limitations of serological typing is that it does not differentiate between many of the alleles that are known to exist in the population. This has prompted the development of methods for analysis of HLA polymorphism at the genetic level, as described in Bidwell, J., Immunology Today 9:18-23, 1988, and Angelini et al., Proc. Natl. Acad. Sci. USA, 83:4489-4493, 1986.
One such method involves the use of DNA restriction fragment length polymorphism (RFLP) as a basis for HLA typing. See Erlich, U.S. Pat. No. 4,582,788, issued Apr. 15, 1986, the contents of which are incorporated herein by reference. RFLP analysis, however, fails to differentiate between certain alleles that are known to exist in the population (e.g., subtypes of HLA-DR4), and thus, cannot be used to distinguish certain combinations of alleles known to exist in the population.
More recently, utilizing the polymerase chain reaction (PCR) process, as described in U.S. Pat. No. 4,683,202, issued Jul. 28, 1987, the contents of which are incorporated herein by reference, researchers have used sequence-specific oligonucleotide ("SSO") probe hybridization to perform HLA-Class II typing. That method entails amplifying a polymorphic region of an HLA locus using the PCR, contacting the amplified DNA to a plurality of sequence-specific oligonucleotide probes under hybridizing conditions, and detecting hybrids formed between the amplified DNA and the sequence-specific oligonucleotide probes. Alleles of HLA-DQ alpha and HLA-DP alpha and beta genes have been identified in the aforementioned manner. See Saiki et al., Nature, 324:163-166, 1986, Bugawan et al., J. Immunol., 141:4024-4030, 1988, and Gyllensten et al., Proc. Natl. Acad. Sci. USA, 85:7652-7656, 1988, the contents of which are incorporated herein by reference.
Reported oligotyping strategies utilizing sequence-specific oligonucleotide probes have failed, however, to resolve many heterozygotic allelic combinations. For example, Fernandes et al., did not resolve all HLA-DRB1 alleles associated with HLA-DRw52 haplotypes due to the sharing of polymorphic sequences within this group of alleles. See Fernandez et al., Hum Immunol 28:51, 1990, the contents of which are incorporated herein by reference. Similarly, Molkentin et al. failed to fully resolve HLA-DQB1-03 alleles using sequence-specific oligonucleotide probe hybridization. Molkentin et al., Hum. Immunol., 31:114, 1991, the contents of which are incorporated herein by reference.
The present invention addresses some of the shortcomings of known HLA typing methods and provides an improved process for resolving HLA alleles and for resolving heterozygotic allelic combinations.